This invention relates to a method and an associated apparatus for automatically determining positions of antibiotic-containing disks in a microbiological testing procedure.
Agar disk diffusion is a widely recognized microbiological assay for measuring susceptibilityxe2x80x94a parameter effectively defined by the assay itself. The susceptibility of a microorganism to a given antibiotic is essentially a description of the size of the inhibitory zone resulting from placement of a permeable disk impregnated with the given antibiotic onto an agar surface inoculated with a sample culture of the microorganism. This parameter provides a measure of the ability of the antibiotic compound to stem growth of the target culture, but it is also a complex function of diffusion constants and other kinetic factors.
Of additional use to the clinician is a related quantitative measure of susceptibility, known as xe2x80x9cminimum inhibitory concentrationxe2x80x9d(MIC). Although still requiring additional information to translate the parameter into a prescription for clinical practice, this quantitative measure eliminates some sources of complexity and uncertainty relative to qualitative susceptibility. A additional useful clinical parameter is the xe2x80x9cinhibitory quotientxe2x80x9d, which expresses the ratio of the drug concentration in a particular body tissue at a lowest clinical dose to the minimum inhibitory concentration.
The MIC is ideally determined by an assay appropriately called the dilution method, which straightforwardly involves inoculating a series of test tubes with the target culture, the test tubes containing a series of dilutions of the target antibiotic. One series of test tubes therefore tests only one culture and one antibiotic, in contradistinction to an agar diffusion assay petri dish, which may test a plurality of antibiotics simultaneously with less material and expense. The advantage of the dilution method is that it provides less ambiguously interpretable quantitative results relative to the agar diffusion method, while its disadvantage is primarily its expense, both in materials and labor.
It is therefore desirable to have a device which automatically translates a dimension of an inhibition zone on an agar diffusion assay plate into a more clinically useful quantitative measure of drug-bacterium interaction, such as the MIC. Such a device is disclosed by U.S. Pat. No. 4,701,850. It is further desirable to have a device which automates the process of reading the apposite linear dimension of the inhibition zone, such devices being revealed in subsequent United States patents. The relation of the diameter of the inhibition zone to the MIC for an unknown biological agent is approximated by a linear relation, the parameters for which assumed relation for a particular antibiotic being determined by statistical estimation based on the scatter of data points whose coordinates are inhibition zone diameters and actual minimum inhibitory concentrations determined by dilution assay for a particular microorganism, the relation being assumed linear and being assumed to persist for untested organisms.
Current commercial microbiological assay machines have largely automated the detection or measurement of agar-plate drug diffusion zones. One current automated test plate reader system requires the user to determine and record the positions of all drug disks on agar plates or panels. While inserting a test plate in the reader, the user must align a reference disk as prompted on screen with a mark on the reader drawer (reference disk is user-defined during drug panel setup). The software then searches for all disks and assigns a correct drug name to each disk based on identified orientation of the reference disk. Each plate is searched for disks in an area approximately 24 mm around the usually 6 mm diameter disk position assigned during drug panel setup. Because the drug code printed on all disks is small and difficult to read visually, it would be a significant advantage for the system to determine a reference disk position automatically without having users align it specifically to the drawer mark.
Sometimes a drug disk may be dropped on the plate in the wrong position(s), or the user may have selected the wrong drug panel name containing the wrong drug disk names. It would be another significant advantage for the system to detect if drug disk(s) were misplaced; this would improve the quality control and reliability of the result reading.
One known technology which has been proposed for use in automatic identification of microbiological assay disks is optical character recognition (OCR) technology. This technology requires input of the orientation of the target characters on each disk prior to reading and recognizing them. It is inherently difficult to know character orientation without an orientation mark in addition to the drug code on each disk. Conventional OCR technology is difficult without an orientation mark, but it can be done through extensive computer processing to assess multiple possible orientations of each disk. Providing an orientation mark on each disk is a solution limited in its usefulness insofar as it requires the supply of special disks in substitution for conventional commercially available disks.
An object of the present invention is to provide an improved apparatus and/or associated method for use in microbiological testing.
A more specific object of the present invention is to provide a microbiological testing apparatus and/or method wherein positions of antibiotic-containing disks are automatically determined.
A related object of the present invention is to provide a microbiological testing apparatus and/or method wherein the zone surrounding a given antibiotic disk is associated with the subject antibiotic compound without further operator intervention or opportunity for human error.
Yet another related object of the present invention is to provide a microbiological testing apparatus and/or method which does not require orientation marks on the disks and the extensive computer processing required by conventional OCR to read codes without that mark.
It is a supplemental object of the present invention to provide such a microbiological testing apparatus and/or method which is inexpensive to use.
A further object of the present invention is to provide an essentially automated microbiological testing apparatus and/or method wherein a dimension of an inhibition zone associated with an antibiotic impregnated disk on an agar plate is measured automatically.
A more particular object of the present invention is to provide such a microbiological testing apparatus and/or method which facilitates association of a microorganism susceptibility measurement with a particular antibiotic agent.
These and other objects of the present invention will be apparent from the descriptions and illustrations provided herein.
A method in accordance with the present invention is adapted for use in a microbiological assay procedure wherein a plurality of diffusion disks are placed on a nutrient medium on a plate, each of the disks carrying a respective antibiotic agent and each having an identification code identifying the respective antibiotic agent, the disks being positioned on the plate in a preselected relative arrangement. The method comprises optically scanning the disks on the plate, consequently generating a digitally encoded image including digitized representations of the identification codes, and electronically processing the digitally encoded image to determine an angle of rotation of the plate relative to a pre-established reference frame (coordinate system), wherein the electronic processing of the digitally encoded image includes detecting a region of the plate occupied by a unique subset of the disks.
The unique subset of the disks may include an absence of a disk in a location within the region, or a pattern or configuration of disks in the region. The unique subset of the disks may alternatively include a reference disk having a unique characteristic relative to other disks on the plate. The unique characteristic may be the area of the respective identification code on the reference disk, an ordered pair of ratio of length and width of the respective identification code, or an ordered pair or ratio of light and dark pixels within the respective identification code. Generally, it is contemplated that the length and width parameters, as well and the light and dark pixels, are measured or counted after the establishing of an outline for the respective identification code.
Where the unique reference characteristic is area, the electronic processing of the digitally encoded image includes (1) digitally processing the digitally encoded image to determine estimates of areas occupied by the identification codes on the disks, and (2) electronically comparing the determined area estimates with prestored identification code areas to automatically distinguish the identification code of the reference disk from among the identification codes of the other disks on the plate.
Where the unique reference characteristic is the length and width combination (e.g., ratio) of a drug carrying disk, the electronic processing of the digitally encoded image includes (1) digitally processing the digitally encoded image to measure lengths and widths of the identification codes of the disks, and (2) electronically comparing the measured lengths and widths with premeasured and electronically stored identification code lengths and widths to automatically distinguish the identification code of the reference disk from among the identification codes of the other disks on the plate.
Where the unique reference characteristic is an ordered pair or ratio of the numbers of light and dark pixels in an identification code, the electronic processing of the digitally encoded image includes (1) digitally processing the digitally encoded image to derive outlines of the identification codes on the disks, (2) further processing the digitally encoded image to count light pixels and dark pixels within the derived outlines of the identification codes on the disks, (3) computing, for each of the disks, a ratio of the respective counted light pixels and dark pixels, and (4) electronically comparing the computed ratios with stored identification code light/dark ratios to automatically distinguish the identification code of the reference disk from among the identification codes of the other disks on the plate.
The present invention is also directed to an associated method comprising optically scanning a plurality of drug diffusion disks on an agar plate and the drug identification codes on the disks, generating a digitally encoded image including digitized representations of the identification codes, and digitally processing the digitally encoded image to determine, for at least one of the identification codes on the disks, a numerical value of at least one overall pattern parameter. The determined numerical value of the pattern parameter is electronically compared with electronically stored premeasured values of the pattern parameter for the identification codes on the disks, to automatically identify at least one disk from among the disks on the plate.
In accordance with another feature of the present invention, the pattern parameter taken from the group consisting of (a) a total identification code area, (b) an ordered pair of identification code length and width, and (c) an ordered pair of light and dark identification code areas.
Where the disks have a preselected relative arrangement on the plate and the one disk constitutes a reference disk, the method further comprises electronically storing a characterization or identification of the preselected arrangement of the disks, the characterization or identification including for each disk a respective antibiotic agent identification code and a location relative to other disks in the arrangement. The method also comprises automatically or electronically determining the identities of the antibiotic agents on the disks in accordance with the preselected arrangement and the reference disk.
In most cases of machine operation, numerical pattern parameter values are determined for each of a plurality of identification codes on the disks. At least some of the determined numerical values of the pattern parameter are electronically compared with electronically stored premeasured values of the pattern parameter associated with the different disks on the plate.
In accordance with another feature of the present invention, the method additionally comprises optically detecting microbe-growth inhibition zones arising about the diffusion disks after placement of the disks in contact with the nutrient medium, electronically determining diameters of the inhibition zones, and automatically computing drug concentration parameters for the antibiotic agents on the disks from the determined diameters of the inhibition zones.
The scanning of the disks is preferably undertaken by a digital camera after growth of a microbe on the microbe-growth medium on the plate.
In accordance with a further feature of the present invention, the electronic processing of the digitally encoded image includes executing an adaptive threshold method to locate the identification codes on the disks and further includes carrying out a 2-bin fast histogram classification of all disks to determine an optimal threshold for distinguishing between relatively light pixels and relatively dark pixels of the identification codes and background areas on the disks.
In accordance with an additional feature of the present invention, the electronic processing of the digitally encoded image further includes filtering the digitally encoded image to eliminate details and simplify the image. The filtering of the digitally encoded image may include digitally implementing a dilation and erosion operation.
In accordance with yet another feature of the present invention, the electronic processing of the digitally encoded image additionally includes determining an outline or code shape of the filtered image of each of the identification codes on the disks. Where the outlines or code shapes of the identification codes of the disks have respective lengths and widths, the electronic processing of the digitally encoded image also includes (i) digitally processing the outlines or code shapes of the identification codes of the disks to measure lengths and widths of the identification codes of the disks, and (ii) electronically comparing the measured lengths and widths with premeasured and electronically stored identification code lengths and widths to automatically distinguish the identification codes of the reference disk from among the identification codes of the other disks on the plate.
Pursuant to a further feature of the present invention, the electronic processing of the digitally encoded image also includes (a) applying an affine transformation to rotate identification code patterns (e.g., individual pixels or identification code outlines), and (b) comparing the rotated identification code patterns with pre-established code shapes. The comparing of the rotated identification code patterns with pre-established code shapes preferably includes computing a 2-dimensional correlation coefficient.
An associated apparatus in accordance with the present invention is for use in carrying out a microbiological assay utilizing a plurality of drug diffusion disks each carrying a respective antibiotic agent and each having an identification code identifying the respective antibiotic agent, wherein the disks are placed in contact with a nutrient medium on a plate and a microbial solution is deposited on the medium. The apparatus comprises an optical monitoring device for optically detecting microbe-growth inhibition zones arising about the diffusion disks after placement of the disks in contact with the nutrient medium and the deposition of the microbial solution on the medium and for optically scanning the identification codes on the disks to produce a digitally encoded image including digitized representations of the identification codes. The apparatus further comprises a processing system operatively connected to the optical monitoring device, the processing system being programmed to digitally process the digitally encoded image to determine, for at least one of the identification codes on the disks, a numerical value of at least one overall pattern parameter. The processing system is additionally programmed to electronically compare the determined numerical value of the pattern parameter with electronically stored premeasured values of the pattern parameter for the identification codes on the disks, to automatically identify at least one disk from among the disks on the plate.
In another aspect of the present invention, the processing system is programmed to digitally process the digitally encoded image to determine an angle of rotation of the plate relative to a pre-established reference frame (coordinate system), the processing system being additionally programmed to detect a region of the plate occupied by a unique subset of the disks.
In a further aspect of the present invention, the processing system is further programmed to identify the antibiotic agents on the disks in response to optically scanned information from the monitoring device, the processing system being also programmed to determine the respective diameters of the microbe-growth inhibition zones.
In an additional aspect of the present invention, the processing system includes generic integrated circuits modified by programming to determine at least one of (a) total identification code area, (b) an ordered pair of identification code length and width, and (c) an ordered pair of light and dark identification code areas.
In yet another aspect of the present invention, the disks have a preselected relative arrangement on the plate and the one disk constitutes a reference disk. The apparatus then further comprises a memory electronically storing a characterization or identification of the preselected arrangement of the disks, the characterization or identification including for each disk a respective antibiotic agent identification code and a location relative to other disks in the arrangement, the processing system including generic integrated circuits modified by programming to automatically or electronically determine the identities of the antibiotic agents on the disks in accordance with the preselected arrangement and the reference disk.
In accordance with another feature of the present invention, the processing system includes generic integrated circuits modified by programming to recognize microbe-growth inhibition zones arising about the diffusion disks after placement of the disks in contact with the nutrient medium, to electronically determine diameters of the inhibition zones, and, from the determined diameters of the inhibition zones, to automatically compute drug concentration parameters for the antibiotic agents on the disks.
In a microbiological testing apparatus and/or method in accordance with the instant invention, positions of antibiotic containing disks are automatically determined, without further operator intervention or opportunity for human error. The apparatus and method do not require orientation marks on the disks. Nor do the apparatus and method necessitate extensive computer processing as required by conventional OCR to read codes without an orientation mark. Thus, the microbiological testing apparatus and method of the invention are inexpensive to use.